Pharmaceutical composition for treating and/or preventing a pathology associated with an obsessional behavior or with obesity

ABSTRACT

This invention relates to the use of a ligand of the 5-HT 4  receptor or of a pharmaceutically acceptable salt of this ligand and to a nucleic acid coding for a 5-HT 4  receptor or of a functionally equivalent receptor for a drug for treating and/or preventing a pathology associated with an obsessional behavior such as anorexia, bulimia and the addiction to drugs of abuse or obesity. The invention also relates to a method for identifying a compound that is biologically active in the treatment and/or the prevention of a pathology associated with an obsessional conduct or obesity including: a) placing the 5-HT 4  receptor or a functionally equivalent receptor in contact with this biologically active compound, and b) the determination of whether this biologically active compound is capable of modulating the basal activity of the 5-HT 4  receptor or of a functionally equivalent receptor.

RELATED APPLICATION

This is a continuation of International Application No.PCT/FR2003/003262, with an international filing date of Oct. 31, 2003(WO 2004/042063, published May 21, 2004), which is based on FrenchPatent Application No. 02/13725, filed Oct. 31, 2003.

FIELD OF THE INVENTION

This invention relates to the treatment and prevention of diseasesassociated with obsessional behaviors such as anorexia, bulimia andaddiction to drugs of abuse, associated or not with stress as well asthe treatment and prevention of obesity. The invention relates moreparticularly to a ligand of the 5-HT₄ receptor for a drug for thetreatment and/or prevention of diseases associated with obsessionalbehaviors and obesity.

BACKGROUND

Food problems coexist with anxiety and depression and represent anincreasing concern of developed countries (Garrow, 1991; Kuczmarski etal., 1994). As for the majority of neurophychiatric pathologies, thecombination of the influence of environmental factors and a geneticpredisposition seems to be responsible for these behavioral deficiencies(Fairburn et al., 1998; Lilenfeld et al., 1998; Barsh et al., 2000). Themothers of 75% of anorexic women suffer from depression or arealcoholics one year before the expression of the symptoms of theirchild. The anorexic syndrome is also detected more frequently in one andthe same family than in the general population without any geneassociated with this pathology having been identified. Bulimicbehaviors, frequently concomitant with anorexia in the same individual,are characterized by impulsive and repeated phases of ingestion of anelevated amount of food.

Bulimia is thus classified among behaviors associated with addiction(international definitions of psychiatry, DSM). Food can be consideredas a reward the obtention of which is based on will (wanting:appetite/incentive motivation) and is motivated by a componentassociated with hedonism (liking: pleasure/palatability) (Hoebel, 1997;Salamone et al., 1997; Stratford and Kelley, 1997; Stratford and Kelley,1999). The excess or the absence of ingesting food is limited not onlyto metabolic and/or endocrinal deficiencies, but also depends on stress(Donohoe, 1984; Morley et al., 1983; Vergoni and Bertoline, 2000), onanxiety (Godart et al., 2000) and on depression (Viesselman and Roig,1985; Casper, 1998).

The hypothalamus, the amygdala and the hippocampus are involved in theregulation of the consumption of food. Moreover, the activity of theneurons of the nucleus accumbens is modified during the anticipation orafter the obtention of a classic reward such as food or drugs of abuse(Di Chiara, 1995; Hoebel, 1997; Koob and Nestler, 1997; Salamone et al.,1997).

Even though the emergence of discoveries shows the involvement ofnumerous peptides in the regulation of food behaviors (leptine,orexines, hypocretines, CART, NPY, POMC, CRH, TRH), the influence ofclassic neuromediators such as serotonin (5-HT) and dopamine (DA) cannotbe avoided. GABA and glutamate should also be considered (Taber andFibiger, 1997; Kelley and Swanson, 1997; Stratford and Kelley, 1997;Stratford et al., 1998).

Dopaminergic systems of the nucleus accumbens are involved in theanticipation of a reward (drugs of abuse, food). A chronicadministration of clozapine, antagonist of the receptors of DA, inducesa hyperphagia (Ackerman and Nolan, 1998; Allison et al., 1999). Cocaineand amphetamine, known for increasing the transmission of DA, areanorexigenic (Foltin and Evans, 1999).

Nevertheless, the serotoninergic systems remain an inevitable link thatcontrols ingestion of food (Barnes and Sharp, 1999) due to the use offenfluramine, inhibitor of the capture of serotonin in obese patients(Guy-B. Grand, 1995).

In sum, deficiencies of the combinations of interactions between factorsof the environment (stress) and genetic factors (genes coding forreceptors present in the brain) appear to be responsible for behavioralproblems such as bulimia, anorexia or the addiction to drugs of abuse.These pathologies and, in a more evident manner, bulimia are consideredtoday to be an addictive behavior.

On the neurobiological level, the state of our knowledge favors thecombined intervention of several neuronal systems for regulating foodbehavior. The best known are the serotoninergic systems that express thecerebral messenger (neuromediator), that is 5-HT. The cerebral areaswhere their actions are manifested are primarily the hypothalamus, theamygdala and the nucleus accumbens.

The exact relationship between the effects of stress and 5-HT isrendered complex by the reciprocal influence between activities of theserotoninergic systems and the hypothalamo-pituitary axis (F. Chaouloff,2000). On the other hand, application of stress brings about increasesin the serotoninergic transmission.

Stress causes elevations in serotoninergic transmission. Theexperimental paradigms in which stresses associated with a conditionedfear bring about an increase in the metabolism and release of 5-HT inthe median pre-frontal cortex (Adell et al., 1997; Inoue et al., 1994),the nucleus accumbens (Inoue et al., 1994; Ge et al., 1997), theamygdala (Amat et al., 1998) and the dorsal hippocampus (Ge et al.,1997; Joseph and Kennett, 1983). In particular, the stress of constraint(forced immobilization) increases renewal of 5-HT in the hypothalamusand the amygdala of the rat and mouse (Konstandi et al., 2000). In thesame manner, the action of corticotropin-releasing hormone or factor(CRF) on the serotoninergic neurons of the corticomesolimbic systemmight be able to modify the rates of 5-HT (Lowry et al., 2000; Price andLucki, 2001). In addition, alterations of the functioning of thereceptors of the glucocorticoids produce variations in the concentrationof 5-HT in the nucleus accumbens (Sillaber et al., 1998). Moreover, therepeated injection of corticosterone increases the activation of theneurons of the hippocampus (CA1) induced by an agonist of the 5-HT₄receptor (Zahorodna et al., 2000). Finally, numerous studies supposethat CRF is responsible for the anorexigenic effect of stress. Inparticular, the intracerebroventricular injection of CRF induces adiminution of ingestion of food in the mouse (Momose et al., 1999).

Serotonin inhibits ingestion of food. The pharmacological approachescombined with the strategies of transgenesis indicate that the receptors5-HT_(1A/1B) and 5-HT_(2A/2C) are involved in regulating ingestion offood and, moreover, of stress (Bonasera and Tecot, 2000; Bouwknecht etal., 2001; Dourish et al., 1986; Heisler et al., 1998; Lucas et al.,1998; Samanin and Garattini, 1996). Anorexia associated with stress isassumed to result from the increase in the activity of serotoninergicneurons. Numerous studies attribute the anorexigenic effect offenfluramine to the activation of the 5-HT_(1B) receptor whereas that ofthe 5-HT_(1A) (autoreceptor), inhibiting the release of 5-HT, induces anelevation of ingestion of food. The insensitivity of mice lackingreceptor 5-HT_(1B) in the injection of fenfluramine confirms itsinvolvement in regulating ingestion of food (Lucas et al., 1998). Thereceptors 5-HT_(2C) also intervene in the consumption of food becausethe mice deprived of it are obese (Heisler et al., 1998). Leptine isknown to reduce ingestion of food, but it is not associated with thisobesity (Nonogaki et al., 1998).

A recent study shows that the 5-HT_(2C) receptors are also responsiblefor the anorexigenic effect of fenfluramine (Vickers et al., 2001).Finally, administration of tropisetrone, antagonist of the receptors5-HT₃ and 5-HT₄, increase ingestion of food of a diet modified by asingle amino acid (Erecius et al., 1996). However, this effect has beenattributed to the 5-HT₃ receptor (Jiang and Gietzen, 1994).Consequently, no data is currently available concerning the contributionof the receptor 5-HT₄ in ingestion of food.

In sum, the current hypothesis for explaining that stress reducesingestion of food is based on two series of parallel studies. The firstone describes that stress increases the activity of thehypothalamo-pituitary axis (stress axis) and the serotoninergic neurons.Furthermore, hyperactivity of the hypothalamo-pituitary axis causes anincrease in the rates of hormones such as CRF, urocortin and, in thefinal stage, of corticosterone. The second series of analyses shows thatthe hormones of the stress axis and 5-HT inhibit the taking of food.

As a consequence, numerous people propose the following sequence ofevents:

Application  of  stress ⇒ brain ⇒ anorex hypothalamo-pituitaryand/or?serotoninergic  neurons

The set of the receptors of 5-HT are coupled with the G proteins withthe exception of the receptor 5-HT₃, that is an ionic channel (Saudouand Hen, 1994). The receptors 5-HT_(1A), 5-HT_(1B), 5-HT_(1D), 5-HT_(1E)and 5-HT_(1F) are negatively coupled to adenylate cyclase and have astrong affinity for 5-HT. Activation of receptors 5-HT₂ stimulatesactivity of phospholipase C (5-HT_(2A/2C)). The other receptors of 5-HTare positively coupled to adenylate cyclase and include 5-HT₄,5-HT_(drol) in the vinegar fly, and the receptors 5-HT₆ and 5-HT₇ inmammals. The receptor 5-HT₄ was described for the first time in thecolliculi (Dumuis et al., 1988) and its stimulation brings about anelevation of the rates of AMPc in the hippocampus, the cerebral cortex,the atrium and the esophagus. In humans, nine subtypes of 5-HT₄receptors named 5-HT_(4A), 5-HT_(4B), 5-HT_(4C), 5-HT_(4D), 5-HT_(4E),5-HT_(4F), 5-HT_(4G), 5-HT_(4H) and 5-HT_(4N) differ by their C-terminalend point (Bockaert et al., 2003, in the press, for review). The systemfor the transduction of receptors 5-HT_(5A) is positively associatedwith adenylate cyclase. That of 5-HT_(5B) has not yet been identified.

Functional influences of the 5-HT₄ receptors have been studiedintensively in the gastrointestinal tract, but little data is availableabout their contributions in the brain. Of the set of the structures ofthe encephalon in rodents and in man, the greatest densities of the5-HT₄ receptor are detected in the limbic system (Waeber et al., 1994).In particular, its concentration is three times greater in the shellthan in the core of the nucleus accumbens (Compan et al., 1996). In thebrain of rodents their rate varies during development and does notattain their adult level until the 21^(st) day after birth (Waeber etal., 1994). In the encephalon of the rat the rates of mRNA's coding forthe 5-HT₄ receptor are greatest in the olfactory system, the striatum,the nucleus accumbens, the habenula and the hippocampus (Gerald et al.,1995; Ulmer et al., 1996; Vilaro et al., 1996).

The agonists of 5-HT₄ receptors cause a reduction in the deficiencies ofmemorization and improve learning by setting transmission ofacetylcholine in motion (J. Bockaert et al., 1998). It is tempting tosuppose that the 5-HT₄ receptor can participate in the neuronalmechanisms of the nucleus accumbens associated with the learning offood. Four pharmacological studies have demonstrated a low contributionof the 5-HT₄ receptor in the “anxiety” state of the rat and of the mouse(Cheng et al., 1994; Silvesre et al., 1996; Kennett et al., 1997;Costall and Naylor, 1997). Inhibition of the 5-HT₄ receptor brings abouta decrease in locomotive activity in the rat under basal conditions(Fontana et al., 1997), in the young mouse 20 to 27 days old (Semenovaand Ticku, 1992) and can attenuate cocaine-induced hyperlocomotion(McMahon and Cunningham, 1999).

In the striatum, serotoninergic control of the rates of extracellular DAby the activation of the 5-HT₄ receptor is simultaneously described asexciting or inhibiting (Bonhomme et al., 1995; Steward et al., 1996;Deurwaerdere et al., 1997).

Finally, stimulation of the 5-HT₄ receptor brings about a closing of theionic channels of potassium (Bockaert ea, 1998), which is capable ofmaintaining the excitability of neurons and augmenting the release ofneuromediators. In agreement with this data, stimulation of the 5-HT₄receptors leads to an increase in the rates of extracellular 5-HT in thehippocampus.

In sum, at the neurobiological level, 5-HT₄ receptors are known tointervene in learning and memory. The possible contribution in motorbehavior and the anxiety state is currently described as moderate andhas been little studied. A single study indicates that this receptor canintervene in the effect of cocaine on locomotive activity.

Furthermore, WO 97/29739 discloses use of antagonists of the 5-HT₄receptor for preparation of a drug intended to avoid, alleviate,suppress or master the gastrointestinal effects caused by a selectiveinhibitor of the re-assimilation of serotonin.

WO 02/11766 discloses use of antagonists of the 5-HT₄ receptor in theprophylaxis or treatment of certain cardiovascular conditions.

SUMMARY OF THE INVENTION

This invention relates to a method of treating and/or preventing apathology associated with an obsessional behavior and/or obesityincluding administering a therapeutically effective amount of a ligandof the 5-HT₄ receptor or of a pharmaceutically acceptable salt of thisligand to a mammal.

This invention also relates to a method of treating and/or preventing apathology associated with an obsessional behavior and/or obesityincluding administering a therapeutically effective amount of a nucleicacid coding for a functional 5-HT₄ receptor or a functionally equivalentreceptor to a mammal.

This invention further relates to a method of identifying a biologicallyactive compound that can be used in a treatment and/or prevention of apathology associated with an obsessional behavior and/or obesityincluding a) contacting a 5-HT₄ receptor or a functionally equivalentreceptor contacting the biologically active compound, and b) determiningwhether the biologically active compound is capable of modulating basalactivity of the 5-HT₄ receptor or of a functionally equivalent receptor.

BRIEF DESCRIPTION OF THE DRAWINGS

Other advantages and characteristics of the invention will appear fromthe following examples concerning the comparative study of mice disabledfor the gene coding for the 5-HT₄ receptor of serotonin and of wild miceand in which reference is made to the attached drawings in which:

FIG. 1 is a schematic representation of objectives of the technical artsput in place to obtain transgenic mice.

FIG. 2 represents the disabling of the gene coding for the 5-HT₄receptor of serotonin. FIG. 2A is a schematic representation of the dDNAfragment (6.5 kb) coding for transmembrane domains II and III of the5-HT₄ receptor of serotonin (exon III). The dDNA sequence (P) representsthe external probe for hybridizing a fragment of dDNA Ase I of 4 kb inwild mice. FIG. 2B is a schematic representation of the cloning vector.The expression of the gene coding for neomycine phosphotransferase (Neo)is under the control of a promoter (phosphoglyderate kinase I promoter:pGK). It was inserted into an enzymatic restriction site Xba in the dDNAsequence coding for the transmembrane domain III of the 5-HT₄ receptor.The size of the fragment of dDNA Ase I that can then be hybridized bythe external probe is 3.5 kb and allows the identification of themutated DNA. FIG. 2C is a Southern blot of the genomic DNA, of embryonicstem cells, previously digested by using the enzyme AseI and hybridizedby the external probe (P).

FIG. 3 shows that the mice disabled for the gene coding for the 5-HT₄receptor of serotonin present a slightly increased weight gain during ashort period of their development. The weight gain is the differencebetween the weights measured on a given day of the development (21 to56) and that of the 20th day after birth. The data are themeans±standard deviation from the average of the variations in theweight gain expressed in g. per day. The rate is measured starting fromgroups constituted by an average number of 21 wild ones (+/+) and 15mutants (−/−) for each day born in one year between the 20th daypreceding weaning and the 56th day of the acquisition of their fertilityafter their birth. Statistical analysis indicated that the weight gainsof the mutant mice were significantly higher than those of the wildanimals only on the days: 27 (+16%, F_(1,72)=6,54; n=40+/+, n=34−/−), 28(+12%, F_(1,67)=4, 73: n=42+/+, n=27−/−), 29 (+30%, F_(1,72)=6, 54;n=23+/+, n=18−/−), 30 (+21%, F_(1,72)=6, 54; n=20+/+, n=19−/−), 36(+12%, F_(1,40)=4, 06; n=22+/+, n=20−/−) and 37 (+13%, F_(1,40)=6, 18;n=25+/+, n=17−/−). No significant difference was detected between thetwo genotypes for the other days. The significant differences betweenthe genotypes are marked by * p<0.05, *** p<0.001.

FIG. 4 shows that the mice disabled for the gene coding for the 5-HT₄receptor of serotonin show abnormal reactions for the taking of food andfor weight gain after the stress of constraint or a forcedimmobilization. FIGS. 4 a and 4 b show the daily taking of food for thewild mice (FIG. 4 a) and the mutants (FIG. 4 b). The daily taking offood is the difference between the quantity of food evaluated during twoconsecutive days. The data is evaluated during a period of habituation(8 days) and of recovery (10 days). The sharp strain of constraint of110 min is applied on the 8^(th) day (arrow). This forced immobilizationinduced a decrease of the taking of food in the wild mice during twodays of the recovery period (FIG. 4 a).

This anorexigenic capacity is less efficacious in the mice lacking the5-HT₄ receptor (FIG. 4 b). FIGS. 4 c and 4 d show the daily variationsof the weight gain in wild mice (FIG. 4 c) and in mutants (FIG. 4 c)from the first day of isolation (9:00 of the illuminated part of thecycle) of the habituation period. The stress of constraint causedsignificant decreases in the weight gain in the wild mice (c) but muchless or not at all in the mutant mice (d). The data is the averages ±the standard deviation from the average (g./day) of groups ofnon-stressed (n=14) or constrained (n=16) wild mice and of mutantanimals without stress (n=17) or immobilized (n=16). The significantdifferences between the stressed animals or not-stressed animals arenoted by §§§ p<0.0001; §§ p<0.001; § p<0.05. The significant differencesbetween the two genotypes are marked by * p<0.05 and the significantinteractions genotype x stress By p<0.05.

FIG. 5 shows that the mice disabled for the gene coding for the 5-HT₄receptor of serotonin consume more food than the wild animals after ananorexigenic stress of increasing intensity. In other words, anexperimental paradigm in which the situation becomes progressively morestressful induced a decrease of the taking of food in the wild micewhereas the anorexigenic effect was reduced in the absence of the genecoding for the 5-HT₄ receptor. The data represent the average ± thestandard deviation from the average of the taking of food (g.) inexperimental contexts that are new and/or adverse for the rodents(Procedure 1: Isolation of 3 h, Procedure 2: Superelevated crosslabyrinth (EPM, 5 min) and isolation of 3 h, Procedure 3: Isolation of96 h, small incision of the tail, injection of NaCl and, EPM). Thesignificant differences between the mice lacking the 5-HT₄ receptor andthe wild animals are marked by * p<0.05. The significant differencesbetween each of the procedures are noted by § p<0.05; §§ p<0.01.

FIG. 6 shows that the mice lacking the gene coding for the 5-HT₄receptor of serotonin proved to be less sensitive to the anorexigeniceffects of MDMA or Ecstasy 24 after the deprivation of food. Thisresistance to the anorexigenic stress of Ecstasy is reproduced if it isconjointly administered with an antagonist selective for the 5-HT₄receptor of serotonin (RS 39604). In other words, the sharp attenuationof the motivation to consume foods induced by the injection of Ecstasyin spite of a depravation of food for 24 h is counteracted by theabsence or the inhibition of the 5-HT₄ receptors. The data represent theaverage ± the standard deviation from the average of the retaking offood (g.) measured 30 min (FIG. 6A), 1 h (FIG. 6B) and 3 h (FIG. 6C)after the injection of NaCl, MDMA (10 mg/kg) and/or of RS39604 (0.5mg/kg) in wild mice (+/+) or mice deprived of the 5-HT₄ receptor (−/−).*p<0.05 significant effect of the genotype after a variance analysisfollowed by test F of Scheffé. § p<0.05; §§§ p<0.001; §§§§ p<0.0001significant effect of the treatment by comparison with the animals ofthe same genotype treated by NaCl (Anova, test F of Scheffé). # p<0.05;p<0.01 significant effect of the treatment by comparison with the wildanimals treated with MDMA (Anova, test F of Scheffé).

FIG. 7 shows that the mice disabled for the gene coding for the 5-HT₄receptor of serotonin present deficiencies of locomotion and/or ofadaptation to a new environment, here the open field. The datarepresents the average of the distance traversed (cm) by the wild mice(+/+, n=7) and the mutants (−/−, n=7) on the total surface (FIGS. 7 aand 7 b) or in the center 99 (FIGS. 7 c and 7 d) of the open fieldduring 30 min and during three consecutive days (day 1 to 3). The dataof FIG. 7 e represents the average time passed in the center of the openfield during three consecutive days. FIG. 7 f represents the averagedata of the vertical activity during three consecutive days. Thesignificant differences between the genotypes are marked by *p<0.05 andbetween the different days of exposure by §§ p<0.01 and § p<0.05.

FIG. 8 shows that the mice disabled for the gene coding for the 5-HT₄receptor of serotonin are less reactive to the superelevated crosslabyrinth (EPM) (FIG. 8 a) whereas in a context of super-induced stressthe mutant mice approved to be more anxious (FIG. 8 b). In the EPMwithout other added stress the mice remain for less time in the openarms (FIG. 8 a) and were less reactive than the control animals becausethe numbers of entries in all the compartments of the EPM are verysignificantly reduced in the absence of the 5-HT₄ receptor (average ±the standard deviation from the average, n=8-9 by genotype, reproducedthree times in different laboratories). In a context of super-inducedstresses the mutant mice began to react to the new environment anddisplayed a more anxious behavior than their wild congeners because theyremained and entered less frequently in the open arms (average ± thestandard deviation from the average, n=18-19 by genotype). Thesignificant differences between the genotypes are marked by *p<0.05(Anova).

FIG. 9 shows that the absence of the gene coding for the 5-HT₄ receptorof serotonin induced a reduction of the rates of leptine. The datarepresents the average ± the standard deviation from the average of theleptine rates of the plasma expressed in ng/mL after the administrationof NaCl (0.9%) or of MDMA (10 mg/kg) in the wild (+/+) or the disabled(−/−) mice for the gene coding for the 5-HT₄ receptor, aged six to 10months on the average. **p<0.01 significant effect of the genotype aftera variance analysis (ANOVA) followed by an F test of Scheffé.

DETAILED DESCRIPTION

I have surprisingly discovered that the absence of the gene coding forthe 5-HT₄ receptor of serotonin renders adult mice less sensitive toanorexigenic stress than wild animals from the embryonic period. Inother words, certain stresses diminish ingestion of food in wild mice,but are less efficacious in mice lacking of the 5-HT₄ receptor. In theabsence of this receptor, mice thus consume more food thannon-genetically modified congeners.

Since stress contributes to the appearance of anorexia, accompaniesbulimia and raises sensitivity to drugs of abuse, I discovered that theligands of the 5-HT₄ receptor can attenuate manifestation of thesepathologies (anorexia, bulimia, addiction to drugs of abuse associatedor not with stress).

The term “5-HT₄ receptor” hereinafter denotes any one of the subtypes(or splicing variants) of the 5-HT₄ receptor such as, for example, thereceptors 5-HT_(4A), 5-HT_(4B), 5-HT_(4C), 5-HT_(4D), 5-HT_(4E),5-HT_(4F), 5-HT_(4G), 5-HT_(4H) and 5-HT_(4N).

The invention therefore relates to the use of a ligand of the 5-HT₄receptor or of a pharmaceutically acceptable salt of this ligand for adrug for treating and/or preventing a pathology associated with anobsessional behavior and/or obesity.

The term “a pathology associated with an obsessional behavior”hereinafter denotes the pathologies involving food problems and, inparticular, anorexia, bulimia and addiction to drugs of abuse associatedor not with stress.

The ligand of the 5-HT₄ receptor used in the framework of the inventionfor a drug for treating and/or preventing a pathology associated with anobsessional behavior and/or obesity is preferably not a ligand of the5-HT₃ receptor and is a specific ligand of the 5-HT₄ receptor.

Inasmuch as I have demonstrated that the 5-HT₄ receptor is involved inthe anorexigenic effective stress, it is now possible to use an agonistof this receptor in a drug intended to treat and/or prevent bulimiawhereas an antagonist or also an inverse agonist of this receptor can beused for a drug intended to treat and/or prevent pathologies associatedwith an obsessional behavior selected from the group constituted byanorexia and addiction to drugs of abuse.

The following terms hereinafter have the following meanings:

-   -   Agonist: Any molecule capable of engendering by its linkage to        its receptors a biological response similar to an endogenic        mediator.    -   Antagonist: Any molecule capable of inhibiting the action of        agonists.    -   Inverse agonist: Any molecule capable of inhibiting the        intrinsic (or basal) activity of the receptor.

All the agonists, all the inverse agonists and all the antagonists,whether known or not yet identified, specific to the 5-HT₄ receptor maybe used in this invention.

Bockaert et al. (1997) described the chemical structure of agonists andof antagonists of the 5-HT₄ receptor. The agonists described belong to 6chemical classes that are the indoles, the benzamides, benzoate, thearyl cetones, the benzimidazolones and the 1,8-naphthalimides. Theantagonists described belong to 5 chemical classes that are thecarboxylates of indole, imidazolpydridine, the benzoates, the arylcetones, the benzimidazolones and the 1,8 naphthalimides. These agonistsand antagonists can be used in accordance with aspects of thisinvention. The inverse agonists described in Claeysen et al., (2001) andin Joubert et al. (2002) can also be used in accordance with aspects ofthis invention.

Moreover, WO 97/29739, WO 02/11766 and WO 02/36113, respectively,disclose antagonists and agonists of the 5-HT₄ receptor, that can alsobe used in accordance with aspects of this invention.

The agonists that can be used herein are selected from the groupcomprising metoclopramide, HTF919(3-(5-methoxy_(—)1H-indole-3-ylmethylene)-N-pentylcarbazimidamidehydrogen maleate), LS650155, BRL 20627, BRL 24682, BRL 24924, cisapride(Carlsson et al., 1997), ML 1035(4-amino-5-chloro-2-[2-(methylsulfinyl)-ethoxy]-N-[(diethylamino)ethyl]benzamidehydrochloride), mosapride (Carlsson et al., 1997), R076186, renzapride,RS 67506 (1-(4-amino-5-chloro-2-methoxyphenyl)-3-[1-(2-methylsulphonylamino)ethyl-4-piperidinyl]-1-propanone hydrochloride),cinitapride, SB 205149, SC 49518(N-[exo-(hexahydro-1H-pyrrolizine-1-yl)methyl]-2-methoxy-4-amino-5-chlorobenzamideHCl), SC 52491, SC53116(4-amino-5-chloro-N-[(hexahydro-1H-pyrrolizin-1-yl)methyl]2-methoxybenzamide),SDZ 216454, TKS 159(4-amino-5-chloro-2-methoxy-N-[(2S,4S)-1-ethyl-2-hydroxymethyl-4-pyrrolidinyl]benzamide,Y 34959, YM 09151(N-(1-benzyl-2-methylpyrrolidine-3-yl)-5-chloro-2-methoxy-4-methylaminobenzamide,YM 47813, zacopride(4-amino-5-chloro-2-methoxy-N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide),ML 10302 (2-piperidinoethyl 4-amino-5-chloro-2-methoxybenzoate), RS57639, SR 49768(2-[(3S)-3-hydroxypiperidino]ethyl-4-amino-5-chloro-2-methoxybenzoate),ADR 932, prucalopride (R093877;4-amino-5-chloro-2,3-dihydro-N-[1-(3-methoxypropyl)-4-piperidinyl]-7-benzofurancarboxamide monohydrochloride), SK951, RS67333(1-(4-amino-5-cloro-2-methoxyphenyl)-3-(1-n-burtl-4-piperidinyl)-1-propanone),RS 17017, RS 56532, YM 53389, BIMU1(3-ethyl-2,3-dihydro-N-[endo-8-methyl-8-azabicyclo(3.2.1)oct-3-yl]-2-oxo-1H-[benzimidazole-1-carboxamide),BIMU8(endo-N-8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3-dehydro-2-oxo-3-(prop-2-yl)-1H-benzimid-azole-1-carboxamide),DAU 6215 ((3-alpha-tropanyl 1H-benzimidazolone-3-carboxamide chloride),DAU 6236, 5-methoxytryptamine, 2-methylserotonin and5-hydroxy-N,N-di-methyltryptamine and 5-carboxamidotryptamine.

The antagonists that can be used are advantageously selected from thegroup comprising tropisetron (ICS 205 930; [(3atropanyl)-1H-indole-3-carboxylic acid ester]), RS 100235(1-(8-amino-7-chloro-1,4-benzodioxan-5-yl)-3-[[3,4-dimethoxyphenyl)prop-1-yl]piperidine-4-yl]propan-1-one, RS 39606, A-85380(3-(2(S)-azetidinylmethoxy)pyridine), GR 113808(1-[2-(methylsulphonyl)amino]ethyl]-4-piperidinyl]methyl1-methyl-1H-indole-3-carboxylate), GR 125487(1-[2-(methylsulphonyl)amino]ethyl]-4-piperidinyl]methyl5-fluoro-2-methoxy-1H-indole-3-carboxylate), GR 138897([1-[2-[methylsulphonyl)amino]-4-piperidinyl]methyl[2-(3-methyl-1,2,4-oxadiazon-5-yl)phenyl]carbamate,SB 203186(1-piperidinyl)ethyl 1H-indole 3-carboxylate), SDZ 205-5572-methyox-4-amino-5-chlorobenzoic acid 2-(diethylamino) ethyl ester,hydrochloride, LY 353433 (1,(1-methylethyl)-N-(2-(4-((tricyclo[2-(3.3.1.1^(3,7)]dec-1-ylcarbonyl)amino-1-piperidinyl)ethyl)-1H-indazole-3-carboxamide),LY 297582, RS 23597(3-piperidine-1-yl)propyl-4-amino-5-chloro-2-methoxybenzoatehydrochloride, SB 204070 (1-butyl-4-piperidinyl)methyl8-amino-7-chloro1,4-benzodioxan-5-carboxylate), DAU 6285((endo-6-methoxy-8-methyl-8-azabicyclo[3.2.1]oct3-yl)-2,3-dihydro-2-oxo-1H-benzimidazole-1 carboxylate hydrochloride),SC53606(1-S,8-S)—N-[hexahydro-1H-pyrrolizin-1-yl)methyl]-6-chloroimidazo[1,2-a]pyridine-8-carboxamidehydrochloride), SC56184, RS67532 (1-(4-amino-5-chloro-2-(3,5-dimethoxybenzyloxyphenyl)-5-(1-piperidinyl)-1-pentanone), GR 125487(1-[2(methylsulfonyl)amino[]ethyl]-4-piperidinyl]methyl-5-fluoro-2-methoxy-1H-indole-3-carboxylatehydrochloride), SB 207078, SB 207266(N-[1-^(n)butyl-4-piperidinyl)methyl]-3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-10-carboxamide),RS 39604(1-[4-amino-5-chloro-2-(3,5-dimethoxyphenyl)methyloxy]-3-[1[2-methylsulphonylamino]ethyl]piperridine-4-yl]propan-1-one,RS 1003002(N-2-(4-(3-(8-amino-7-chloro-2,3-dihydro-1,4-benzodioxin-5-yl)-3-oxopropyl)pyperidin-1-yl) ethyl)-methanesulfonamide), ML 10375(2-cis-3,5-dimethylpiperidino) ethyl 4-amino-5-chloro2 methoxybenzoate),SB 207710(1-butyl-4-piperidinyl)methyl-8-amino-7-1,4-benzodioxan-5-carboxylate),SB205800(N-(1-butyl-4-piperidinyl)methyl-8-amino-7-chloro-1,4-benzodioxan-5-carboxamide),N 3389, FR 1052 and R 50595.

The inverse agonists that can be used herein are selected from the groupcomprising RO 116-2617, RO 116-0086 and RO 116-1148.

The amount of ligand of the 5-HT₄ receptor or of a pharmaceuticallyacceptable salt of this ligand that is effective in the treatment and/orprevention of a pathology associated with an obsessional behavior and/orobesity depends on the nature of the disorder and can be readilydetermined by known experimental techniques and clinical standards.Furthermore, simple in vitro trials can optionally be used to identifythe ranges of optimal dosages. The effective dosages can be readilyextrapolated from dosage-response curves obtained from the in vitro testsystems or from the animal model.

The pharmaceutical compositions for treating and/or preventing apathology associated with an obsessional behavior and/or obesitycomprising a ligand of the 5-HT₄ receptor are prepared in accordancewith standard pharmaceutical practices. These pharmaceuticalcompositions are presented in a form appropriate for a parenteraladministration, oral, rectal, nasal, transdermic, pulmonary, central orsystemic administration or the like.

The pharmaceutical compositions contain in addition to the ligand of the5-HT₄ receptor or the pharmaceutically acceptable salt of this ligand atleast one pharmaceutically acceptable vehicle selected as a function ofthe administration path and of the form of administration.

In another aspect, the invention relates to restitution in targetedareas of the brain of a “wild” 5-HT₄ receptor for treating or preventingpathologies associated with obsessional behaviors and/or obesity and, inparticular, pathologies associated with obsessional behaviors with thehypothesis that these pathologies are caused by one or several mutationsin the gene coding for the 5-HT₄ receptor.

Consequently, the invention relates to the use of a nucleic acid codingfor a functional 5-HT₄ receptor or a functionally equivalent receptorfor the preparation of a drug for treating and/or preventing a pathologyassociated with an obsessional behavior.

The term “functional 5-HT₄ receptor” hereinafter denotes a wild 5-HT₄receptor present in normal pharmacological properties.

The term “functionally equivalent receptor” hereinafter denotes areceptor whose amino acid sequence is close to that of the 5-HT₄receptor (at least 90% identical and preferably at least 95% identical)and is activated by the same agonists and with the same intensity as thewild 5-HT₄ receptor.

The molecule of nucleic acid coding for a functional 5-HT₄ receptor or afunctionally equivalent receptor is advantageously a molecule coding fora mammalian 5-HT₄ receptor and, in particular, a molecule coding for ahuman 5-HT₄ receptor. These sequences can be obtained in Genbank underthe numbers:

For the human: Y09586 (5-HT_(4A)), Y10437 (5-HT_(4B)), Y12506(5-HT_(4C)), Y15507 (5-HT_(4D)), AJ011371 (5-HT_(4G));

For the mouse: Y09587 (5-HT_(4A)), Y09585 (5-HT_(4B)), Y09588(5-HT_(4E)), AJ011370 (5-HT_(4F));

For the rat: U20906 (5-HT_(4A)), 020907 (5-HT_(4B)) and AJ011371(5-HT_(4E));

For the guinea pig: Y13585 (5-HT_(4B)).

As a result of degeneration of the genetic code, other nucleic acidsequences coding for substantially the same amino acids can be used inthe invention. The sequences are by way of example and in anon-exhaustive manner nucleotide sequences comprising all or part of thenucleic acid coding for a 5-HT₄ receptor modified at the level of one orseveral codons in such a manner as to produce a silent mutation. Thesequences of nucleic acid that can be used in the invention can beobtained by various methods known in the art, e.g., via cDNA obtainedfrom the mRNA of the 5-HT₄ receptor after a reverse transcription.

In an advantageous manner the nucleus accumbens, the amygdale, thehippocampus and the hypothalamus or the cerebral structures in which thesimultaneous restoration of the expression of the gene coding for the5-HT₄ receptor and the removal of the deficiencies in the regulation ofthe taking of food associated with this absence are envisioned. Only asimultaneous restoration of the molecular and cellular deficiencies(rate of the endogenic monoamines, activity of the serotoninergicneurons, for example) and behavioral deficiencies as a consequence ofthe reestablishing the expression of this gene permit a validation of acausal connection between the molecular and behavioral phenotype.

To advantageously express a functional 5-HT₄ receptor or a functionallyequivalent receptor in the nucleus accumbens, the amygdale, thehippocampus and the hypothalamus the use of transfer vectors is possiblesuch as non-viral or viral vectors containing nucleic acid moleculescoding for a functional 5-HT₄ receptor or a functionally equivalentreceptor.

Among the viral vectors that can be used in the invention, virusesassociated with the adenovirus of type 2, vectors stemming from thelentivirus with different pseudotypes, vectors stemming from viruses offeline immunodeficiency and vectors stemming from the foamy virus, amongothers, are possible. The viral vectors should be disencumbered of anypathogenic and/or toxic effect.

The expression of the molecule of nucleic acid coding for a functional5-HT₄ receptor or a functionally equivalent receptor contained in thesevectors is advantageously placed under the control of adequate promotersas a function of the target tissue. For the expression of a functional5-HT₄ receptor or a functionally equivalent receptor in the nucleusaccumbens, the amygdale, the hippocampus and/or the hypothalamus, anadvantageously used promoter can be the promoter of the gene coding forthe transporter for capturing dopamine for the nucleus accumbens.

The invention relates in another aspect to a method of identifying abiologically active compound that can be used in the treatment and/orprevention of a pathology associated with an obsessional behavior and/orobesity and in particular a biologically active compound for thetreatment and/or prevention of a pathology associated with anobsessional behavior comprising the following steps:

a) placing the 5-HT₄ receptor or a functionally equivalent receptor incontact with this biologically active compound, and

b) determining whether this biologically active compound is capable ofmodulating the basal activity of the 5-HT₄ receptor or of a functionallyequivalent receptor.

The term “biologically active compound” hereinafter denotes any naturalor synthetic chemical compound capable of attenuating the symptoms of apathology associated with obsessional behaviors after itsadministration.

Stage (a) of the method can comprise the following steps:

i) placing cells in a culture that express a functional 5-HT₄ receptoror a functional equipment receptor, and

ii) incubation of the cells with this biologically active compound.

The terms “functional 5-HT₄ receptor” and “a functionally equivalentreceptor” are previously defined.

The cells cultivated in stage (i) of the method are advantageously cellsthat overexpress a functional 5-HT₄ receptor or a functionallyequivalent receptor on their surface. Every cell capable ofoverexpressing a functional 5-HT₄ receptor or a functionally equivalentreceptor on its surface can be used herein. The human embryonic cellsHEK 293 can be cited as examples and in a non-exhaustive manner.

A molecule of nucleic acid coding for a functional 5-HT₄ receptor or afunctionally equivalent receptor can be used to transform the cells thatoverexpress a functional 5-HT₄ receptor or a functionally equivalentreceptor.

The vector for transforming cells overexpressing a functional 5-HT₄receptor or a functionally equivalent receptor can comprise at least onemolecule of nucleic acid coding for a functional 5-HT₄ receptor or afunctionally equivalent receptor like the one described above,advantageously associated with control sequences adapted to the processof the expression or of the production of these receptors in a cellularhost. The vector used is selected as a function of the host (cultivatedcells) in which it is to be transferred; this can be any vector such asa plasmid. Preparation of these vectors as well as production orexpression of the receptor in a cellular host can be realized bytechniques of molecular biology and of genetic engineering well-known inthe art.

A compound capable of modulating the base activity of a receptor iseither an agonist, an inverse agonist or an antagonist of this receptor.Linkage of an agonist, an inverse agonist or an antagonist to a receptorbrings about changes in the conformation of this receptor and in thecell a transduction of the signal by the intermediation of secondmessengers is observed. Consequently, step (b) measures by any adaptedmeans the affinity between the receptor and the biologically activecompound after they have been brought into contact.

Modulation of the basal activity of the 5-HT₄ receptor or of afunctionally equivalent receptor can be advantageously measured viaactivation (for an agonist) or inhibition (for an antagonist or for aninverse antagonist) of the transduction of the signal from the 5-HT₄receptor. One skilled in the art, knowing the cascade of events inducedduring transduction of the signal from this receptor, is capable ofdetermining adequate methods and conditions for measuring the activationor the inhibition of the transduction of the signal from the 5-HT₄receptor, e.g., by measuring the quantity of cAMP before and after theplacing in contact with the functionally active compound.

In another aspect of the method stages (a) and (b) can be realized byfixing one or several 5-HT₄ receptors or functionally equivalentreceptors on one or several membranes. The 5-HT₄ receptors or thefunctionally equivalent receptors can also be integrated into abiosensor. In such a system it is possible to visualize in real time theinteractions between the compound to be tested and the receptor. One ofthe partners of the receptor/ligand couple is fixed on an interface thatcan contain a matrix covered with aliphatic chains. This hydrophobicmatrix can be readily covered by a lipidic layer by the spontaneousfusion of liposomes injected at its contact. The 5-HT₄ receptors orfunctionally equivalent receptors inserted in the liposomes can then beintegrated into the biosensors. The biologically active compound is thenanalyzed relative to one or several 5-HT₄ receptors or functionallyequivalent receptors. In addition, the technology of the biosensorallows the affinity of the linkage to be measured.

The invention also relates to a method for treating and/or preventing apathology associated with an obsessional behavior comprisingadministering an efficacious quantity of a ligand of 5-HT₄ receptorssuch as previously defined or of a nucleic acid coding for a 5-HT₄receptor or functionally equivalent receptor such as previously defined.

The results obtained herein show that the 5-HT₄ receptors control theleptine rates and can, by virtue of this fact, intervene in the controlof obesity. In fact, I have shown that the absence of 5-HT₄ receptorsinduces a reduction of the leptine rates. This involvement in obesity ispossible because obesity has been observed in mutant mice deprived ofthe 5-HT₄ receptor more than 6 months old and therefore older than thoseused in the experimental part below.

Consequently, this invention relates to the use of a ligand of the 5-HT₄receptor such as it was previously defined and in particular an agonistof the 5-HT₄ receptor such as it was previously defined for apharmaceutical composition for the treatment and/or prevention ofobesity. All embodiments envisaged for the pharmaceutical compositionsfor the treatment and/or prevention of obsessional behaviors apply tothe treatment and/or prevention of obesity.

In a particular aspect, the invention relates to the restitution intargeted areas of the brain of a “wild” 5-HT₄ receptor for treating orpreventing obesity with the belief that this pathology is caused by oneor several mutations in the gene coding for the 5-HT₄ receptor.

Consequently, the invention relates to the use of a nucleic acid codingfor a functional 5-HT₄ receptor or a functionally equivalent receptorfor a drug for treating and/or preventing obesity. The variousembodiments envisaged above (mammalian 5-HT₄ receptor, human 5-HT₄receptor, viral or non-viral transfer vector and the like) also apply tothe treatment and the prevention of obesity.

Thus, the invention provides a method for the treatment and/orprevention of obesity comprising administering an efficacious quantityof a ligand and, in particular, an agonist of the 5-HT₄ receptors suchas they are defined above or of the nucleic acid coding for a functional5-HT₄ receptor or a functionally equivalent receptor such as definedabove.

The invention relates to a method for identifying a biologically activecompound capable of being used in the treatment and/or the prevention ofobesity and, in particular, a biologically active compound for thetreatment and/or the prevention of obesity comprising the followingsteps:

a) placing the 5-HT₄ receptor or a functionally equivalent receptor incontact with this biologically active compound, and

b) determination of whether this biologically active compound is capableof modulating the basal activity of the 5-HT₄ receptor or of afunctionally equivalent receptor.

Various aspects of the identification of a biologically active moleculein the treatment and/or the prevention of a disease associated with anobsessional behavior apply with such modifications as the circumstancesrequire to the identification of a biologically active molecule in thetreatment and/or the prevention of obesity. Taking into account theresults obtained herein, it is evident to one skilled in the art that amolecule active in the treatment and/or the prevention of obesity shouldbehave like an agonist of the 5-HT₄ receptor.

I. MATERIALS AND METHODS I.1. Generation of Mice Disabled for the GeneCoding for the 5-HT₄ Receptor of Serotonin

a. Principle

Transgenesis is a general principle and comprises modification of theexpression of a gene of interest in a living organism. In the mousethese modifications require three successive steps (FIG. 1). The firststep uses a large number of techniques of molecular biology. It consistsof the obtention of a genomic construction resulting from deletion orinsertion of the gene of interest or also from the modification bydirected mutagenesis of several nitrogenous bases. The second step usestransfection of embryonic cells by transfection of the genomicconstruction in totipotent, murine embryonic cells (“embryonic stemcells” or ES). Insertion of the mutated gene into their genome isbrought about by random homologous recombination in a stable manner. Therecombinant clones are then selected by using an antibiotic (G418 orneomicine) because a resistance gene was previously inserted during therealization of the genomic construction. The last stage is the injectionof the positive clones into the blastocytes (FIG. 2).

b. Verification of the Absence of the 5-HT₄ Receptors in the Mutant Mice

To verify the absence of the 5-HT₄ receptors in the mutant mice thetechnique of radiography was used as previously described (Compan etal., 1996) from cerebral sections of the wild animals, heterozygotes andmutants. The linkage sites of the 5-HT₄ receptor have been marked by aselective radioligand, the [³H]GR113808 (Amersham), antagonist of the5-HT₄ receptor.

In the wild mice a heterogeneous distribution of the linkage sitesmarked by the [³H]GR113808 was observed in the base ganglia, the limbicsystem or the hippocampal formation has already observed for the rat((Waeber et al., 1994, Compan et al., 1996). No specific marking wasdetected in the mutant mice. The density of the marked linkage sites wasreduced in all the cerebral structures analyzed in the heterozygotousmice in comparison to the wild mice (Table 1).

TABLE 1 Density of the linkage sites of the 5-HT₄ receptor marked by the[³H]GR113808 (0.1 nM) in adult mice of the wild and of theheterozygotous genotype. B_(max) (average ± standard deviation from theAverage in fmol/mg of protein Regions Wild Heterozygotous Olfactorytubercles 201 ± 34 66 ± 16 (67%) Ventral pallidum 171 ± 37 53 ± 21 (70%)Nucleus accumbens 116 ± 28 36 ± 16 (68%) Rostral striatum 115 ± 15 48 ±11 (48%) Caudal Striatum 142 ± 25 18 ± 10 (85%) Globus pallidus  90 ± 2014 ± 8 (85%) Hippocampus 104 ± 11 9 ± 8 (91%) * Considering aconcentration of 1 mg of protein/10 mg of cerebral tissue.

The genotype of the previously identified was also verified by using thetechnique of polymerization in a chain reaction (touchdown protocolPCR). All the animals tested were born from heterozygotous couples andaged between 4 and 6 months. We obtained 18% of the mutant mice over aperiod of three years. We still do not have an explanation about thevalue of this report, which does not follow the Mendelian laws (Table2).

TABLE 2 Mendelian relationships obtained after the crossing ofheterozygotous mice Wild Heterozygotous Homozygotous 36% (414) 46% (632)18% (209)

The mice deprived of the 5-HT₄ receptors develop without apparentproblems with the exception of a weight gain that is significantly moreelevated than that of the wild mice over a short period of theirdevelopment (FIG. 3). No significant difference of weight was detectedin the mice aged from 4 to 6 months (not illustrated). Since classicmechanisms of adaptation of the neuronal systems can occur during thecourse of development, the hypothesis according to which an unaccustomedcontext could induce problems in the consumption of food during theiradult period was formed by the inventor.

I.2. Behavioral Tests

The consumption of food and weight gain of the mutant mice in comparisonto the wild animals were therefore measured after the constraint test(forced immobilization) had been used, which is known for constitutingan anorexigenic stress. In the same manner, ingestion of food wasevaluated after the using of conflict tests: The superelevated crosslabyrinth and the open field. In these two experimental tests therodents were confronted with a conflict between the motivation toexplore a new environment and the fear of open and/or superelevatedspaces.

a. Animals

The wild and mutant animals stemmed from the line Sv 129/Ter (Phillipset al., 1999) and were born from crossings of heterozygotous mice (+/−)for the mutation of the gene coding for the 5-HT₄ receptor to retain thesame genetic predisposition between the two genotypes. The animals wereraised and manipulated under conditions of standard illumination at thecontrolled and constant temperature and degrees of hydrometry of 22° C.and 55% relative humidity. A day/night cycle (12/12 h) was artificiallymaintained. The food was in the form of cylindrical croquettes (23%proteins; 3.5% raw fatty matter; 3% raw cellulose; 7.5% raw ash; 12%humidity). The genotpye of the mice was identified by using thetechnique of polymerase chain reaction or PCF. All the experiments wereperformed with animals with an average age of 4 to 6 months.

b. Evaluation of Ingestion of Food

After the constraint test. Each of the tests was divided into threephases: A period of habituation (7 days), the day of the constraintstress (immobilization for 110 min) and a recovery stage (10 days). Themice were divided into a control group and a constraint group. On thestress day the wild mice disabled for the gene coding for the 5-HT₄receptor received an injection of NaCl 9%, 1 mg/kg of RS 39604,antagonist of the 5-HT₄ receptor. In parallel thereto, a group of micedid not receive any treatment and were weighed like the previous miceand immobilized, if necessary, 10 minutes after the start of theirmanipulation. Ingestion of food was then measured 2 h 30 min, 3 and 5 hand each day during the recovery period.

After an isolation of 3 h (procedure 1). The animals are isolated in acage in the presence of food and water ad libitum. The weight of thefood and of the animal was evaluated before and 3 h after isolation. Thedifference between two successive weights revealed the quantity of foodingested by the mouse. As the bottom of the cage was a grill, it waspossible to evaluate the quantity of food wasted by each animal.

After the superelevated cross labyrinth (procedure 2: moderate stress).The same animals were regrouped four to a cage for one week in aresidence chamber. On the test day, after a period of 30 min the animalswere placed in the superelevated cross labyrinth and then isolated toevaluate their consumption of food 3 h after the beginning of the test.

After the superelevated cross labyrinth anticipated by an incision(procedure 3: high stress). During a period of habituation each mousewas isolated for four days (96 hours) in the presence of food and watersupplied ad libitum. The weight of the mice and of the food consumed wasmeasured daily at the same hour to establish a baseline. On the fourthday a small incision on the tail was made. A blood sample was then takenfor 10 min to analyze the plasmatic rates of corticosterone before theinjection and the conflict test. The superelevated cross labyrinth alsorepresents a stress inductor because it induces an increase of the ratesof corticosterone (Rodgers et al., 1999). Incisions were made in adifferent room of the test area and all useful precautions were taken toavoid any stress aside from the incision of the tail and theimmobilization prior to this operation. The blood samples obtained werecentrifuged 10 min at 10,000 rpm and the plasma then stored at −80° C.until the further dosages. The corticosterone was dosed using thetechnique of radioimmunoassay (ICN Clinisciences). Once the sample wasconcluded the animals were placed in the test area.

On the fifth day the animals received an intraperitoneal injection(i.p.) of NaCl at 9. The injections were made 10 min before the micewere placed in the superelevated cross labyrinth. The animals weredivided into two test groups of wild mice treated with NaCl (n=8) andmutants that also received an injection of NaCl (n=12). The mice wereplaced in the center of the superelevated cross labyrinth 10 min afterthe injection. A small incision on the tail was again made 30 min afterthe test, in which the rates of corticosterone reached their maximum andremained elevated for four hours (Natelson et al., 1987). After thetest, two successive evaluations of the taking of food were carried out3 h after the start of the test.

After the open field. We proceeded in the same manner as in the case ofprocedure 2 to evaluate ingestion of food 3 h after the beginning of theconfrontation with the open field. Furthermore, motor activity of themice was evaluated one month after procedures 2 and 3.

After the administration of drugs. Each drug used to treat the wild micedisabled for the gene coding for the 5-HT₄ receptor was dilutedextemporaneously in a saline solution of NaCl (0.9%) and injected in asystemic manner (i.p.). The injection volume of each treatment was 200μL for 30 g. The wild mice or those disabled for the 5-HT₄ receptor(n=35) received the following treatments: NaCl, MDMA (or ecstasy or3,4-N-methylene dioxymethamphetamine, SIGMA, 10 mg/kg), RS 39604, RS102221/MDMA.

The MDMA (10 mg/kg, Sigma, user license 9900431 S, V. Compan) and RS39604 (0.5 mg/kg), antagonist of the 5-HT₄ receptor, were administeredby themselves or in a combined treatment to the wild mice or thosedeprived of the 5-HT₄ receptor (n=12-17 by genotype and pharmacologicalproducts). The dosage of 0.5 mg/kg for the RS 39604 was selected becauseits administration induced an increase of the taking of food in thenourished wild mice at libitum and remained without effect in the mutantanimals for the 5-HT₄ receptors in comparison to the mice treated withNaCl or with different dosages of RS 39604 (0.01; 0.1; 1 and 10 mg/kg;not illustrated).

Ingestion of food by the wild mice and the mice disabled for the genecoding for the 5-HT₄ receptor was evaluated in accordance with the testprotocol described by Lucas et al., 1998. The different mice wereisolated for a habituation period of three days in the presence of foodand water supplied ad libitum during which the weights of the mice andof the food consumed were measured daily at the same hour. On the fourthday the mice were deprived of their food for 24 hours. The drugs wereinjected such as NaCl after the food deprivation period to determine theeffect of the different treatments on ingestion of food. Food wasreintroduced into each cage after an interval of 10 min for the othertreatments. Three successive weighings of the food were then carried out30 min, 1 h and 3 h after the reintroduction of the food.

c. Conflict Tests

The superelevated cross labyrinth is constituted by two rectangularareas (L: 57 cm, 1:5 cm) that are fixed at a right angle (90°). One ofthe two areas has walls 15 cm high and is called “closed arms”. Theother area, without walls, is called “open arms”. This device is placedon a base at a height 30 cm above the floor. After a habituation periodof 30 min in the test room each animal is placed at the intersection ofthe two areas and filmed for 5 minutes without the testers beingpresent. The analysis of the data consists of evaluating the number ofentries and the time passed in the open arms or the closed arms by themouse, the time passed in the center and the number of times the animalinclined its head toward the floor when it was present in the open arms(head dips).

When they were placed in the test, the mice were face-to-face with theconflict between the exploration of a new environment and the fear ofopen spaces and height. Factorial analyses give evidence of two types ofbehavior: The one associated with anxiety and the other associated withmotor activity (Brunner et al., 1999). It is commonly known that anxiousanimals return more frequently and remain longer in the closed arms andinversely for the open arms because mice are nocturnal animals.

The open field is an area 43.2×43.2 cm whose walls have a height of 30cm. Infrared sensors are arranged on the four sides at a distance of 1.5cm from each other and are thus adapted to the size of the mice. Therecording of the distance traveled is carried out by software (MEDAssociates Activity Monitor) for 30 min starting from the moment atwhich the animal is placed in the center of the test device. Thisprotocol is repeated on three consecutive days. It is thus possible toevaluate the habituation capacity of the mice in this new environment.The testers are not present in the test room during the recording. Theinfrared grid permits a fine analysis of several variables to be made.

II. RESULTS II.1. Hyposensitivity of the Mice Deprives of the 5-HT₄Receptor in Anorexigenic Stress: Constraint Stress or ForcedImmobilization

Constraint stress, proposed as an experimental model for the study ofanorexia (Rybkin et al., 1997, Harris et al., 2002) has been used fortesting the resistance limits of mice disabled for the gene coding forthe 5-HT₄ receptor to not consume food after stress. Ingestion of foodand variations of weight gain were measured for a habituation period of8 days and of a recovery of 10 days (FIG. 4).

During an isolation period of 8 days (habituation period) ingestion offood by the mice lacking the 5-HT₄ receptor was not different than thatof the wild mice. Variations in their weight gain during the habituationperiod were less than those of the wild animals, as is indicated by thesignificant interaction between the genotype and the time(F_(1,360)=2,45; p<0.05). In other words, no significant variation ofweight gain was detected in the mutant mice (F_(39, 186)=0.94) whereasit was increased in the wild mice (F_(29, 176)=4, 45; p<0.001).Ingestion of food or the changes in weight gain between the two groupsof animals of the same genotype, programmed to be immobilized or notimmobilized on the 8^(th) day, were not significantly different (FIG. 4a,b).

After the constraint stress the statistical analysis (repeated Anovameasuring) indicated a significant effect of the stress on ingestion offood over the course of time (_(9,567)=13, 99; p<0.0001) and asignificant genotype x time interaction (F_(9,549)=2.2, p<0.05). Theanalysis (two-way Anova) also revealed that the capacity of theconstraint stress to induce a lowering of ingestion of food depends onthe genotype the first 24 h following the stress (F_(1,61)=6, 73;p<0.05). Consumption of food was significantly reduced in the wild mice(−36.4%, FIG. 4 a) and, in a lesser amplitude, in the mutant mice(−19.6%, FIG. 4 b) in comparison to the non-stressed mice of the samegenotype. A more detailed analysis indicated that the mice deprived ofthe 5-HT₄ receptor consumed significantly more food than the wild mice24 h after the immobilization (+24%, p<0.05). After 48 h only the wildmice consumed even less food relative to the control animals (−16.4%,FIG. 4 a). No significant effect of the stress on ingestion of food bythe mutant mice was detected 48 h after the forced immobilization (FIG.4 b).

In a parallel manner, the statistical analysis (Anova, repeatedmeasurements) indicated a significant effect of the stress on thevariations in the weight gain over the course of time (F_(9,522)=11, 33;p<0.0001) and a significant genotype x time interaction (F_(9,522)=2,38; p<0.05). The statistical analysis (Anova, repeated measurements)revealed that the constraint stress induces a significant lowering ofweight gain in the wild mice (F_(1,252)=5.76, p<0.05, FIG. 4 c) but notin the mice disabled for the gene coding for the receptor(F_(1,270)=0.014, FIG. 4 d). A detailed statistical analysis indicatedthat the stress caused lowerings of weight gain in the wild mice thefirst 4 days of the recovery period in comparison to the control mice(FIG. 4 c). On the other hand, the constraint stress had a lesserefficaciousness in the absence of the 5-HT₄ receptor since their weightgain remained stable and weaker in comparison to their stressedcongeners during 24 h after immobilization (FIG. 4 d).

Moreover, the results indicate that if the constraint stress is precededby an injection of RS39604, antagonist of the 5-HT₄ receptor, after theforced immobilization of wild females, ingestion of food by the wildmice was not modified (not shown). In other words, the pharmacologicalinactivation of the 5-HT₄ receptor suppressed the anorexigenic effect ofthe constraint stress. In a parallel manner, the administration ofRS39604 reduced the weight losses of the wild mice in comparison to theanimals treated with NaCl (not shown) (Compan et al., 2003).

II.2. Hyposensitivity of the Mice Deprived of the 5-HT₄ Receptor Upon anAnorexigenic Stress of Increasing Intensity

The results indicate that the adult mice deprived of the 5-HT₄ receptordisplayed less sensitivity to the anorexigenic effects of newenvironments (FIG. 5). The statistical analysis showed a significanteffect of the genotype (F_(1, 47)=5, 57; p<0.05) and of the proceduresused (F_(1, 47)=18, 55; p<0.0001).

Transferring the mice from their customary cage to the cages for theevaluation of ingestion of food did not modify the consumption of foodby the wild and the mutant mice (procedure 1, FIG. 5). The moderatestress (procedure 2), a single exposure in the superelevated crosslabyrinth for 5 min, known to increase the activity of thehypothalamo-pituitary axis, induced a significant diminution ofingestion of food in the wild mice (−33.4%; p<0.05; n=7). This was notthe case for the mutant mice (FIG. 5, n=9). The consumption of food bythe mice deprived of the 5-HT₄ receptor was significantly more elevatedthan that of the wild animals after the moderate stress (+40 8, 4%;F_(1,15)=7, 97; p<0.05; n=10; FIG. 5). When using other series ofanimals our results indicate that an intense stress (procedure 3)involves a greater reduction of ingestion of food in the wild mice incomparison to the mice treated with procedure 2 (−70 8%; F_(2, 19)=16,11; p<0.0001; n=8; FIG. 5). Even if ingestion of food also diminishes inthe absence of the 5-HT₄ receptor, the mutant mice consume more foodthan the wild animals after a strong stress (+132%; F_(1,22)=6.7;p=0.0042; FIG. 5; n=10).

II.3. Absence or Inactivation of the 5-HT₄ Receptor CounteractsInhibition of the Motivation to Consume Food Induced by Administrationof MDMA or of Ecstasy

The global statistical analysis of ingestion of food reveals significanteffects of genotype (F_(1,105)=4,86; p<0.05), treatment (F_(1,105)=7.24;p<0.001) and time (F_(3,315)=390,12; p<0.0001). The interactions betweenthe genotype and the treatment (F_(3,105)=2,76; p<0.05) and between thefactors of time and treatment (F_(9,315)=29,30; p<0.0001) are alsosignificant.

In the case of MDMA, the statistical analysis shows the significanteffects of the genotype (F_(1,59)=11.93; p<0.001) and of the treatment(F_(1,59)=15,78; p<0.001) that vary over the course of time(F_(3,177)=3,97; p<0.01) and (F_(3,177)=53,12; p<0.0001), respectively.The results indicate that administration of MDMA induced a significantdiminution of ingestion of food in the wild mice in comparison to therodents of the same genotype treated with NaCl (FIG. 6). This effect wasobserved 30 min (−95,38%, FIG. 6A) and 1 h (−88.56%, FIG. 6B) after itsadministration and is absent at 3 h (FIG. 6C). In the mice lacking the5-HT₄ receptor, compared to the mutant rodents treated with NaCl, MDMAalso induces a significant diminution of ingestion of food 30 min(−69,07%, FIG. 6A) and 1 h (−68,49%, FIG. 6B) after its administration.This effect is no longer observed 3 h after the treatment of the mice(FIG. 6C). Furthermore, consumption of food in mice lacking the 5-HT₄receptor is significantly more elevated 1 h (+645, 56%; p<0.001, FIG.6B), and 3 h (+244, 23%; p<0.05, FIG. 6C) after administration of MDMAin comparison to wild animals that received the same treatment.

For RS 39604, antagonist of the 5-HT₄ receptor, the statistical analysisdoes not reveal significant genotype effects (F_(1, 63)=1, 37) either ofthe treatment (F_(1, 63)=3, 93) or of an interaction between the factorsof genotype and treatment (F_(1, 63)=3, 93) (FIG. 6).

In the case of MDMA combined with RS 39604, selective antagonist of the5-HT₄ receptor, the statistical analysis shows a significant effect ofthe treatment (F_(1, 55)=7, 10; p<0.05) over the course of time(F_(3, 165)=6, 87; p<0.0001). The results indicate that the combinedadministration of MDMA and of RS 39 604 induced a significant diminutionof ingestion of food in the wild mice in comparison to the rodents ofthe same genotype treated with NaCl. This effect was observed 30 min(−83, 72%, FIG. 6A) and 1 h (−64, 89%, FIG. 6B) after administration ofMDMA/RS 39 604. No variation in the consumption of food was observed 3 hafter the treatment of the animals (FIG. 6C). Our results are similar inthe mice lacking the 5-HT₄ receptor. Administration of MDMA/RS 39604significantly reduces their consumption of food 30 min (−82, 19%, FIG.6A) and 1 h (−72, 88%, FIG. 6B) after injection of MDMA/RS 39604 and iswithout effect at 3 h (FIG. 6C) in comparison to mice of the samegenotype. Moreover, the results reveal that ingestion of food by wildmice is significantly more elevated 1 h (+206,82%, FIG. 6B) and 3 h(+51,18%, FIG. 6C) after administration of MDMA/RS 39604 than that ofwild animals that received only MDMA. This effect is not revealed in themutant mice for the 5-HT₄ receptor.

II.4. Reaction to Novelty of the Mutant Mice for the 5-HT₄ Receptor inthe Open Field

The results presented in FIG. 7 show that the mutant mice travelsignificantly less distance in the open field than the wild animals onlythe first day of their exposure (F_(1,14)=6.76, p<0.05). No significantdifference between the two genotypes was detected on the second and thethird day of exposure. The mice deprived of 5-HT₄ receptors alsoremained significantly less time in the center in comparison to theirwild congeners on the first (F_(1,14)=5.50; p<0.05) and the second day(F_(1,14)=4.50, p<0.05) of exposure in the open field, which suggests amore elevated level of anxiety in the mutant mice (FIG. 7 e). Nosignificant difference between the two genotypes was detected for thevertical activity (FIG. 7 f), which indicates that the mutant mice wouldnot present deficiencies of exploratory activity.

This data shows a diminution of the reactivity to novelty and/or of thelocomotive activity of the mice deprived of the 5-HT₄ receptor incomparison to the wild mice.

II.5. Study of the Anxiety Level of the Mice Deprived of the 5-HT₄Receptor

I have taken account of the coexistence of the variations of anxiety andfood problems in bulimic patients and then analyzed whether variationsof the state “of anxiety” of the mice deprived of the 5-HT₄ receptorcould be connected to their persistence in consuming food in spite ofthe application of an anorexigenic stress.

I used the superelevated cross labyrinth for this purpose, constitutedby areas or closed and open arms. The more the rodents pass and enterinto the closed arms the more they are considered as “anxious”. Theprincipal result indicates that procedure 3 (strong stress) induced asignificant elevation of the time spent or of the number of entries intothe closed arms in comparison to procedure 2 (moderate stress) (FIG. 8).Only the number of head dips, which is an index of the exploratoryactivity, significantly diminishes after procedure 3 in comparison toprocedure 2 in the wild mice (FIG. 8). These results suggest that themice deprived of the 5-HT₄ receptor present a more elevated level “ofanxiety” in an adverse situation (procedure 3) relative to the wildmice.

Furthermore, significant diminution of the total number of entries intothe open and closed arms by the mutant mice in the case of procedure 2(labyrinth only) relative to wild mice suggests a lowering of theirlocomotive activity and/or a deficiency in adapting to novelty.

The study of the alimentary behavior under the influence of stress doesnot seem to be able to be envisaged without considering the influence oflocomotive activity. Its increase can be associated with more elevatedenergetic needs and thus with a stronger consumption of food andinversely.

The data obtained during the study of the reaction to novelty (II.4.above) associated with the data obtained by using the superelevatedcross labyrinth suggests that the stimulation of the 5-HT₄ receptor isassociated with increases in locomotive activity and/or a non-adaptedincrease in reactivity faced with new environments. This beliefreinforces that of the involvement of the 5-HT₄ receptor in addictions.

III. DISCUSSION

Three types of molecular mechanisms are capable of conferring aresistance to anorexigenic stresses for mice deprived of the 5-HT₄receptor.

III.1. Inability of Serotoninergic Systems to Adapt to Stress toSufficiently Inhibit Ingestion of Food by Mutant Mice

I then gave special weight to the belief of a deficiency of theserotoninergic transmission after the application of a stress in theabsence of the 5-HT₄ receptor. The forced immobilization caused anelevation of the rates of extracellular 5-hydroxyindol acetic acid(5-HIAA) solely in the wild mice. In other words, the rates of the mainmetabolite of 5-HT remain unchanged in the absence of the 5-HT₄receptor.

The hyposensitivity of the mutant mice to an anorexigenic stress mighttherefore be based on the absence of a modification of the rates ofextracellular 5-HIAA (FIG. 9).

III.2. Augmentation of the Expression of the mRNA's Coding for thePeptide CART in the Mutant Mice

Augmentation of the expression of the mRNA's coding for the peptide CART(cocaine—amphetamine-related transcripts, experimental MDMA paradigm)observed in the mutant mice in the nuclei accumbens and/or in thehypothalamic nuclei in comparison with the wild mice (not shown) permitsthe formulation of the following belief: The set of the ligands of the5-HT₄ receptor is capable of controlling consumption of food by virtueof the fact of the control of the expression of the CART peptide.

Administration of this peptide can induce anorexigenic and orexigenicaffects in accordance with its administration in the lateral ventriclesor in the nuclei of the hypothalamus.

III.3. Lowerings of the Rates of Leptine Detected in the Mutant Mice

Lowerings of the rate of leptine detected in the mice disabled for the5-HT₄ receptor (FIG. 9, experimental MDMA paradigms) allows the proposalthat the ligands of the 5-HT₄ receptors can regulate the variations inthe consumption of food and in weight by the control of the rates ofleptine.

The statistical analysis of the variations of the rates of leptine didnot reveal a significant effect of the treatment with MDMA (p=0.95).These results indicate that a single administration of MDMA does notmodify the rates of leptine in the wild or in the mutant mice (FIG. 9).

In the absence of the gene coding for the 5-HT₄ receptor the statisticalanalysis of the variations of the leptine rates reveals a significantgenotype effect (F_(1,27)=10,42; p<0.01). These results show that theleptine rates are significantly less elevated in the mice disabled forthe gene coding for the 5-HT₄ receptor in comparison to the wild animals(−21%) as the use of test F of Scheffé also indicates (p<0.01) (FIG. 9).

III.4. Conclusion

The anorexigenic effect of stress could be based on a cascade of eventsof which the first link is an increase in the activity of thehypothalamo-pituitary axis. An elevation of the transmission ofmonoamines (5-HT and DA) that inhibits the taking of food follows. Inthe current state of knowledge none of the receptors of 5-HT was shownto represent a molecular element responsible for the anorexigenic effectof stress with the exception of a single pharmacological study on the5-HT_(2A/2C) receptor (Grignaschi et al., 1993). It was quite recentlyreported that the weight of mutant mice for the 5-HT_(2C) receptordiminishes in a manner comparable to that of wild mice as a consequenceof the repeated application of constraint stress (Clifton et al., 2003).

The behavioral results demonstrate a hyposensitivity of the mice lackingthe 5-HT₄ receptor to an anorexigenic stress, confirming the inhibitorycontrol of 5-HT on the consumption of food. They support the hypothesisof the contribution of 5-HT in the anorexigenic effect of stress ofwhich the 5-HT₄ receptor is one of the mediators. Taking into accountthe modifications of the serotoninergic parameters in the NAc and thehypothalamus in the absence of the 5-HT₄ receptor, it is then possiblethat stress does not increase the release of 5-HT in a proportionsufficient to diminish the taking of food of mutant mice after stress.

Some data is in favor of a positive control of the release of the 5-HTas a consequence of the activation of the 5-HT₄ receptor. Thestimulation of the 5-HT₄ receptor induces a closure of the ionicchannels of potassium (Bockaert et al., 1998), which is capable ofmaintaining the excitability of the neurons and increasing the releaseof neuromediators. In agreement with this data, stimulation of the 5-HT₄receptor leads to an elevation of the rates of extracellular 5-HT in thehippocampus (Ge & Barnes, 1997). In the base conditions (without stress)this data shows an elevation of the 5-HIAA/5-HT ratio in thehypothalamus and the NAc, which suggests an alteration of the metabolismof the 5-HT in the absence of the 5-HT₄ receptor. Moreover, theseresults suggest that the lowering of the rates of the 5-HT in thehypothalamus after stress is not compensated in the mice deprived of the5-HT₄ receptor even though the density membrane transporter of the 5-HTmarked by the tritiated citalopram is stronger than in the NRD.

Interestingly, leptine, the genetic disabling of which renders miceobese, can increase the rate of renewal of the 5-HT in the hypothalamus(Calapai et al., 1999; Hastings et al., 2002) as well as theconcentration of the capture transporter of the 5-HT in the frontalcortex (Charnay ea., 1999). Moreover, disabling the gene coding forleptine induces a diminution of the rates of mRNA coding for themembrane transporter of 5-HT in the NRD (Collin et al., 2000).Localization of the receptors of leptine on the serotoninergic neuronsof NRD and the nucleus of the median raph of the mouse (Finn et al.,2001) represents a supplementary argument in favor of an interactionbetween leptine and the serotoninergic neurons. In the same experimentalparadigm of the use of MDMA these results indicate that the rates ofleptine are diminished in the mutant mice. Furthermore, the injection ofMDMA did not induce a variation of leptinemia. This data indicates apositive control of the leptine rates as a result of the activation ofthe 5-HT₄ receptor after a fasting of 24 h in agreement with the risesof leptine induced by the administration of 5-hydroxy tryptophane(Yamada et al., 1999). However, the leptine rates are not modified inthe mutant mice for the 5-HT_(1B) receptor (Bouwknecht et al., 2001) or5-HT_(2C) (Nonogaki et al., 1998). This data indicates that leptinemiais lower in the mice deprived of the 5-HT₄ receptors before and afterthe constraint stress in comparison with the wild mice. Since anelevation of the leptine rates follows that of corticosterone(Guerre-Millo, 1997), these results indicate that the effects of thestress of depriving food on leptinemia are rendered ineffective in theabsence of the 5-HT₄ receptor; the result would be a greater resistanceof the mice disabled for the 5-HT₄ receptor to anorexigenic stresses.

Since leptine activates the expression of the peptide CART in theparaventricular nucleus of the hypothalamus (Kristensen et al., 1998),the possible variations of the peptide CART in the hypothalamus of micedisabled for the 5-HT₄ receptor are being analyzed in comparison to thewild mice. Their involvement in the regulation of the consumption offood is variable in accordance with its injection site. Thus, theintracerebroventricular administration of the peptide CART results inanorexia (Kristensen et al., 1998) whereas an intratissularadministration in the hypothalamus causes hyperphagia (Abbott et al.,2001). The orexigenic effect of an injection of the CART peptide in theparaventricular nucleus of the hypothalamus described by Abbott et al.(2001) is contradicted by the observations of Stanley et al. (2001). Theresults herein indicate that injection of the CART peptide into theshell of the NAc reduced the appetite in the same experimental paradigmof the use of MDMA. The set of the results therefore indicates that MDMAinhibits the appetite by activation of the 5-HT₄ receptor, bringingabout a positive regulation of the expression of the CART peptide andits release. Some data constitutes arguments in favor of such amechanism—(1) the 5-HT₄ receptor is localized on the neurons containingGABA (Compan et al., 1996), (2) the CART peptide is colocalized with theGABA in the neurons of the NAc (Scearce-Levie et al., 1999,Scearce-Levie et al., 2001); the results presented here suggest aregional colocalization of the expression of the mRNA's and of the CARTpeptide proteins/5-HT₄ receptor in the shell of the NAc. However, thecellular colocalization and the estimation of the concentration of thepeptide in the NAc after each of these treatments remains to beestablished.

REFERENCES

-   Abbott C R. Rossi M. Wren A M. Murphy K G. Kennedy A R. Stanley S A.    Zollner A N. Morgan D G. Morgan I. Ghatei M A. Small C J. Bloom    S R. (2001) Endocrinology, 142, 3457-63.-   Ackerman S, Nolan L J. (1998) CNS Drugs., 9, 135-51.-   Adell A. Casanovas J M. Artigas F. (1997) Neuropharmacology, 36,    735-741.-   Allison D B. Mentore J L. Heo M. Chandler L P. Cappelleri J C.    Infante M C. Weiden P J. (1999) J. Psychiatry, 156, 1686-96.-   Amat J. Matus-Amat P. Watkins L R. Maier S F. (1998) Brain Res.,    812, 113-120.-   Barnes N M. & Sharp T. (1999) Neuropharmacology, 38, 1083-152.-   Barsh G S. Farroq I S. O'Rahilly S. (2000) Nature, 404, 644-51.-   Bockaert et al. (1997) Handbook of Experimental Pharmacology,    Vol. 129. Serotonergic Neurons and 5-HT receptors in the CNS.    Eds. H. G. Baumgarten and M. Göthert. Springer-Verlag Berlin    Heidelberg. 439-474.-   Bockaert J. Ansanay H. Letty S. Marchetti-Gauthier E. Roman F.    Rondouin G. Fagni L. Soumireu-Mourat B. Dumuis A. (1998) C R Acad    Sci III., 321(2-3), 217-21.-   Bockaert J. Claeysen S. Sebben M. Dumuis A. (1998) Ann N Y Acad.    Sci., 861, 1-15.-   Bockaert J. Claeysen S. Compan V. Dumuis A. (Revue, 2003, sous    presse) 5-HT₄ receptor in CNS functions: are they potential    therapeutical targets? Drug Target-   Bonasera S J. Tecott L H. (2000) Pharmacol Ther., 88 (2): 133-42.-   Bonhomme N. De Deurwaërdere P. Le Moal M. Spampinato U. (1995)    Neuropharmacology, 3, 4269-279.-   Bouwknecht J A. Van der Gugten J. Hijzen T H. Maes R A. Hen R.    Olivier B. (2001) Psychopharmacology, 153(4), 484-90.-   Brunner D. Buhot, M-C. Hen R. Hofer M. (1999) Behav Neurosci., 113,    587-601.-   Calapai G. Corica F. Corsonello A. Sautebin L. Di Rosa M. Campo G M.    Buemi M. Mauro V N. Caputi A P. (1999) J Clin Invest., 104, 975-82.-   Carlsson L. Amos G J. Andersson B. Drews L. Duker G.    Wadstedt G. (1997) J Pharmacol Exp Ther., 282, 220-7.-   Casper R C. (1998) Depress Anxiety, 8, 96-104.-   Chaouloff F. (2000) J. Psychopharmacol., 14, 139-151.-   Charnay Y. Cusin I. Vallet P G. Muzzin P. Rohner-Jeanrenaud F.    Bouras C. (2000) Neurosci Lett., 283, 89-92.-   Claeysen S. Sebben M. Becamel C. Parmentier M-L. Dumuis A.    Bockaert J. (2001) EMBO reports, 21, 61-67.-   Clifton P G. Lee M D. Somerville E M. Kennett G A. Dourish    C T. (2003) Eur J Neurosci., 17, 185-90.-   Collin M. Hakansson-Ovesjo M. Misane I. Ogren S O. Meister B. (2000)    Brain Res Mol Brain Res., 81, 51-61.-   Compan V. Charnay Y. Daszuta A. Hen R. Bockaert J. (2003) Comptes    rendus de la Société de Biologie de Paris.-   Cheng C H K. Costall B. Kelly M. Naylor R J. (1994) Eur J    Pharmacol., 255, 39-49.-   Compan V. Daszuta A. Salin P. Sebben M. Bockaert J. Dumuis A. (1996)    Eur J Neurosci., 8, 2591-2598.-   Compan V. Dusticier N. Nieoullon A. Daszuta A. (1996) Synapse, 24,    87-96.-   Costall B. & Naylor R J. (1997) Br J Pharamcol., 122, 1105-1118.-   De Deurwaerdère P. L'Hirondel M. Bonhomme N. Lucas G. Cheramy A.    Spampinato U. (1997) J Neurochem., 68, 195-203.-   Di Chiara G. (1995) Drug Alcohol Depend, 38, 95-137.-   Donohoe T P. (1984) Life Science, 34(3), 203-218.-   Dourish C T. Hutson P H. Kennett G A. Curzon G. (1986) Appetite, 7    Suppl, 127-40.-   Dumuis A. Bouhelal R. Sebben M. Cory R. Bockaert J. (1988) Mol.    Pharmacol., 34, 880-887.-   Erecius L F. Dixon K D. Jiang J C. Gietsen D W. (1996) J Nutri.,    126, 1722-1731.-   Fairburn C G. Doll H A. Welch S L. Hay P J. Davies B A. O'Connor    M E. (1998) Arch Gen Psychiatry, 55, 233-241.-   Finn P D. Cunningham M J. Rickard D G. Clifton D K. Steiner    R A. (2001) J Clin Endocrinol Metab., 86, 422-6.-   Foltin R W. & Evans S M. (1999) Pharmacol Biochem Behav., 62,    457-64.-   Fontana D J. Daniels S E. Wong E H. Clark R D. Eglen R M. (1997)    Neuropharmacology, 36, 689-96.-   Garrow J. (1991) Br Med J., 303, 704-706.-   Ge J & Barnes N M. (1996), Br J Pharmacol, 117, 1475-80.-   Ge J. Barnes N M. Costall B. Naylor R J. (1997) Pharmacol Biochem    Behav., 58, 775-783.-   Gerald C. Adham N. Kao H T. Olsen M A. Laz T M. Schetcher L E. Bard    J A. Vaysse P J. Hartig P R. Branchek T A. (1995) EMBO J., 14,    2806-2815.-   Godart N T. Flament M F. Lecrubier Y. Jeammet P. (2000) Eur    Psychiatry., 15, 38-45.-   Grignaschi G. Mantelli B. Samanin R. (1993) Neurosci Lett., 152,    103-6.-   Guerre-Millo M. (1997) Biomed Pharmacother., 51, 318-23.-   Guy-Grand B. (1995) Obes. Res., 4, 491S-496S.-   Harris R B. Mitchell T D. Simpson J. Redmann S M Jr. Youngblood B D.    Ryan D H. (2002) Am J Physiol Regul Integr Comp Physiol. 282,    R77-88.-   Hastings J A. Wiesner G. Lambert G. Morris M J. Head G.    Esler M. (2002) Regul Pept., 103, 67-74.-   Heisler L K. Chu H M. Tecott L H. (1998) Ann N Y Acad. Sci., 15:    861:74-8.-   Hoebel B G. (1997) Appetite, 29, 119-133.-   Inoue T. Tsuchiya K. Koyama T. (1994) Pharmacol. Biochem. Behav.,    49, 911-920.-   Jiang J C. & Gietzen D W. (1994) Pharmacol Biochem Behav., 47,    59-63.-   Joseph M H. & Kennett G A. (1983) Brain Res., 270, 251-257.-   Joubert L. Claeysen S. Sebben M. Bessis A S. Clark R D. Martin R S.    Bockaert J. Dumuis A. (2002) J Biol. Chem., 277, (28) 25502-11.-   Kelley A E. Swanson C J. (1997) Behav Brain Res., 89, 107-113.-   Kennett G A. Bright F. Trail B. Blackburn T P. Sanger G J. (1997)    Neuropharmacology, 36, 707-712.-   Konstandi M. Johnson E. Lang M A. Malamas M. Marselos M. (2000)    Pharmacol Res., 41, 341-346.-   Koob G F. Nestler E J. (1997) J Neuropsychiatry Clin Neurosci., 9,    482-497.-   Kristensen P. et al. (1998) Nature, 393, 72-6.-   Kucksmarski R J. Flegal K M. Campbell S M. Jonhson C L. (1994) J Am    Med. Assoc., 272, 205-211.-   Lilenfeld L R. et al., (1998) Arch Gen Psychiatry, 55, 603-610.-   Lowry C A. Rodda J E. Stafford L. Lightman Ingram C D. (2000) J.    Neurosci., 20, 7728-7736.-   Lucas G. Di Matteo V. De Deurwaerdère P. Porras G. Martin-Ruiz R.    Artigas F. Esposito E. Spampinato U. (2001) Eur. J. Neurosci., 13,    889-890.-   Lucas J. Yamamoto A. Scearce-Levie K. Saudou F. Hen R. (1998) J    Neurosci., 18, 5537-5544.-   Mc Mahon L R. & Cunningham K A. (1999) J Pharmacol Exp Ther., 291,    300-307.-   Momose K. Inui A. Asakawa A. Ueno N. Nakajima M. Fujimiya M.    Kasuga M. (1999) Diabetes Obes Metab., 5, 281-284.-   Morley J E. Levine A S. Rowland N E. (1983) Life Science, 32,    2169-82.-   Nonogaki K. Strack A M. Dallman M F. Tecott L H. (1998) Nat. Med.,    4(10), 1152-6.-   Pelleymounter M A. Cullen M J. Baker M B. Hecht R. Winters D.    Boone T. Collins F (1995) Science, 28; 269(5223), 540-3.-   Phillips T. J. Hen R. Crabbe J. C. (1999) Psychopharmacology 147,    5-7.-   Price M L. Lucki I. (2001) J. Neurosci., 21, 2833-2841.-   Rodgers R J. Haller J. Holmes A. Halasz J. Walton T J. Brain    P F. (1999) Physiol. Behav, 68, 47-53.-   Rybkin I I. Zhou Y. Volaufova J. Smagin G N. Ryan D H. Harris    R B. (1997) Am J Physiol. 273, R1612-22.-   Salamone J D. Cousins M S. Snyder B J. (1997) Neurosci Biobehav    Rev., 21, 341-359.-   Samanin R. Garattini S. (1996) Therapie, 51, 107-15.-   Saudou F. Hen R. (1994) Medical Chemistry Research, 4, 16-84.-   Scearce-Levie K. Viswanathan S S. Hen R. (1999) Psychopharmacology,    141, 154-161.-   Scearce-Levie K. Coward P. Redfern C H. Conklin B R. (2001) Trends    Pharmacol Sci., 22, 414-20.-   Semenova T P. Ticku M K. (1992) Brain Res., 588(2), 229-36.-   Sillaber I. Montkowski A. Landgraf R. Barden N. Holsboer F.    Spanagel R. (1998) Neuroscience, 85, 415-425.-   Silvestre J S. Fernandez A G. Palacios J M. (1996) Eur J Pharmacol,    309, 219-222.-   Stanley S A. Small C J. Murphy K G. Rayes E. Abbott C R. Seal L J.    Morgan D G. Sunter D. Dakin C L. Kim M S. Hunter R. Kuhar M. Ghatei    M A. Bloom S R. (2001) Brain Res., 893, 186-94.-   Steward L J. Ge J. Stowe L R. Brown C. Bruton R K. Stokes P R A.    Barnes N M. (1996) J. Pharm., 117, 55-62.-   Stradford T R. Kelley A. (1997) J Neurosci., 17, 4434-4440.-   Stradford T R. Kelley A. (1999) J Neurosci., 19, 11040-8.-   Stradford T R. Swanwon C J. Kelley A. (1998) Behav Brain Res., 93,    43-50.-   Taber M T. Fibiger H C. (1997) Neuroscience, 76, 1105-112.-   Ulmer C. Engels P. Abdel'Al A. Lubbert H. (1996) Naunyn-Schmied    Arch. Pharmacol., 354, 210-212.-   Vergoni A V. & Bertolini A. (2000) Eur J. Pharmacol., 405 (1-3),    25-32.-   Vickers S P. Dourish C T. Kennett G A. (2001) Neuropharmacology, 2,    200-9.-   Visselman J O. Roig M. (1985) J Clin Psychiatry, 46, 118-24.-   Vilaro M T. Cortès R. Gerald C. Branchek T A. Palacios J M.    Mengod G. (1996) Mol Brain Res., 43, 356-360.-   Waeber C. Sebben M. Nieoullon A. Bockaert J. Dumuis A. (1994)    Neuropharmacology, 33, 527-541.-   Yamada J. Sugimoto Y. Ujikawa M. (1999) Eur J Pharmacol., 383,    49-51.-   Zahorodna A, Tokarski K, Bijak M. (2000) Pol J Pharmacol., 52(2),    107-9.

1-20. (canceled)
 21. A method of treating anorexia in a mammal in needthereof comprising administering a therapeutically effective amount ofan antagonist, partial agonist, or inverse agonist of a 5-HT₄ receptoror of a pharmaceutically acceptable salt of the antagonist, partialagonist, or inverse agonist to the mammal to treat anorexia in themammal.
 22. The method of claim 21, wherein an antagonist isadministered.
 23. The method of claim 22, wherein the antagonist isselected from the group consisting of tropisetron (ICS 205 930; [(3atropanyl)-1H-indole-3-carboxylic acid ester]), RS 100235(1-(8-amino-7-chloro-1,4-benzodioxan-5-yl)-3-[[3,4-dimethoxyphenyl)prop-1-yl]piperidine-4-yl]propan-1-one,RS 39606, A-85380 (3-(2(S)-azetidinylmethoxy)pyridine), GR 113808(1-[2-(methylsulphonyl)amino]ethyl]-4-piperidinyl]methyl1-methyl-1H-indole-3-carboxylate), GR 125487(1-[2-(methylsulphonyl)amino]ethyl]-4-piperidinyl]methyl5-fluoro-2-methoxy-1H-indole-3-carboxylate), GR 138897([1-[2-[methylsulphonyl)amino]-4-piperidinyl]methyl[2-(3-methyl-1,2,4-oxa-diazon-5-yl)phenyl]carbamate,SB 203186 (1-piperidinyl)ethyl 1H-indole 3-carboxylate), SDZ 205-5572-methyox-4-amino-5-chlorobenzoic acid 2-(diethylamino)ethyl ester,hydrochloride, LY 353433(1,(1-methylethyl)-N-(2-(4-((tricyclo[2-(3.3.1.1.sup.3.7]dec-1-ylcarbonyl)am-ino-1-piperidinyl)ethyl)-1H-indazole-3-carboxamide),LY 297582, RS 23597(3-piperidine-1-yl)propyl-4-amino-5-chloro-2-methoxybenzoatehydrochloride, SB 204070 (1-butyl-4-piperidinyl)methyl8-amino-7-chloro1,4-benzodioxan-5-carboxylate), DAU 6285((endo-6-methoxy-8-methyl-8-azabicyclo[3.2.1]oct3-yl)-2,3-dihydro-2-oxo-1-H-benzimidazole-1carboxylate hydrochloride), SC53606(1-S,8-S)—N-[hexahydro-1H-pyrrolizin-1-yl)methyl]-6-chloroimidazo[1,2-a]p-yridine-8-carboxamidehydrochloride), SC56184, RS67532 (1-(4-amino-5-chloro-2-(3,5-dimethoxybenzyloxyphenyl)-5-(1-piperidinyl)-1-pentanone), GR 125487(1-[2(methylsulfonyl)amino[]ethyl]-4-piperidinyl]methyl-5-fluoro-2-methoxy-1H-indole-3-carboxylatehydrochloride), SB 207078, SB 207266(N-[1-.sup.nbutyl-4-piperidinyl)methyl]-3,4-dihydro-2H-[1,3]oxazino[3,2-a-]indole-10-carboxamide),RS 39604(1-[4-amino-5-chloro-2-(3,5-dimethoxyphenyl)methyloxy]-3-[1[2-methylsulphonylamino]ethyl]piperridine-4-yl]propan-1-one,RS 1003002(N-2-(4-(3-(8-amino-7-chloro-2,3-dihydro-1,4-benzodioxin-5-yl)-3-oxopro-p-yl)pyperidin-1-yl)ethyl)-methanesulfonamide),ML 10375 (2-cis-3,5-dimethylpiperidino)ethyl 4-amino-5-chloro2methoxybenzoate), SB 207710(1-butyl-4-piperidinyl)methyl-8-amino-7-1,4-benzodioxan-5-carbo-xylate),SB205800(N-(1-butyl-4-piperidinyl)methyl-8-amino-7-chloro-1,4-benzodioxan-5-carboxamide),N 3389, FR 1052 and R
 50595. 24. The method of claim 21, wherein aninverse agonist is administered.
 25. The method of claim 24, wherein theinverse agonist is selected from the group consisting of RO 116-2617, RO116-0086 and RO 116-1148.
 26. The method of claim 21, wherein a partialagonist is administered.
 27. The method of claim 26, wherein the partialagonist is selected from the group consisting of LS65 0155, ML10302,RS67333, Prucalopride, and Cisapride.
 28. The method of claim 21,wherein the mammal is a human
 29. A method of treating behavior of ananorexic mammal in need thereof comprising administering atherapeutically effective amount of an antagonist, partial agonist, orinverse agonist of a 5-HT₄ receptor or of a pharmaceutically acceptablesalt of the antagonist, partial agonist, or inverse agonist to themammal to treat the mammal.
 30. The method of claim 29, wherein anantagonist is administered.
 31. The method of claim 30, wherein theantagonist is selected from the group consisting of tropisetron (ICS 205930; [(3a tropanyl)-1H-indole-3-carboxylic acid ester]), RS 100235(1-(8-amino-7-chloro-1,4-benzodioxan-5-yl)-3-[[3,4-dimethoxyphenyl)prop-1-yl]piperidine-4-yl]propan-1-one,RS 39606, A-85380 (3-(2(S)-azetidinylmethoxy)pyridine), GR 113808(1-[2-(methylsulphonyl)amino]ethyl]-4-piperidinyl]methyl1-methyl-1H-indole-3-carboxylate), GR 125487(1-[2-(methylsulphonyl)amino]ethyl]-4-piperidinyl]methyl5-fluoro-2-methoxy-1H-indole-3-carboxylate), GR 138897([1-[2-[methylsulphonyl)amino]-4-piperidinyl]methyl[2-(3-methyl-1,2,4-oxa-diazon-5-yl)phenyl]carbamate,SB 203186 (1-piperidinyl)ethyl 1H-indole 3-carboxylate), SDZ 205-5572-methyox-4-amino-5-chlorobenzoic acid 2-(diethylamino)ethyl ester,hydrochloride, LY 353433 (1,(1-methylethyl)-N-(2-(4-((tricyclo[2-(3.3.1.1.sup.3.7]dec-1-ylcarbonyl)am-ino-1-piperidinyl)ethyl)-1H-indazole-3-carboxamide),LY 297582, RS 23597(3-piperidine-1-yl)propyl-4-amino-5-chloro-2-methoxybenzoatehydrochloride, SB 204070 (1-butyl-4-piperidinyl)methyl8-amino-7-chloro1,4-benzodioxan-5-carboxylate), DAU 6285((endo-6-methoxy-8-methyl-8-azabicyclo[3.2.1]oct3-yl)-2,3-dihydro-2-oxo-1-H-benzimidazole-1carboxylate hydrochloride), SC53606(1-S,8-S)—N-[hexahydro-1H-pyrrolizin-1-yl)methyl]-6-chloroimidazo[1,2-a]p-yridine-8-carboxamidehydrochloride), SC56184, RS67532 (1-(4-amino-5-chloro-2-(3,5-dimethoxybenzyloxyphenyl)-5-(1-piperidinyl)-1-pentanone), GR 125487(1-[2(methylsulfonyl)amino[]ethyl]-4-piperidinyl]methyl-5-fluoro-2-methoxy-1H-indole-3-carboxylatehydrochloride), SB 207078, SB 207266(N-[1-.sup.nbutyl-4-piperidinyl)methyl]-3,4-dihydro-2H-[1,3]oxazino[3,2-a-]indole-10-carboxamide),RS 39604(1-[4-amino-5-chloro-2-(3,5-dimethoxyphenyl)methyloxy]-3-[1[2-methylsulphonylamino]ethyl]piperridine-4-yl]propan-1-one,RS 1003002(N-2-(4-(3-(8-amino-7-chloro-2,3-dihydro-1,4-benzodioxin-5-yl)-3-oxopro-p-yl)pyperidin-1-yl)ethyl)-methanesulfonamide),ML 10375 (2-cis-3,5-dimethylpiperidino)ethyl 4-amino-5-chloro2methoxybenzoate), SB 207710(1-butyl-4-piperidinyl)methyl-8-amino-7-1,4-benzodioxan-5-carbo-xylate),SB205800(N-(1-butyl-4-piperidinyl)methyl-8-amino-7-chloro-1,4-benzodioxan-5-carboxamide),N 3389, FR 1052 and R
 50595. 32. The method of claim 29, wherein aninverse agonist is administered.
 33. The method of claim 32, wherein theinverse agonist is selected from the group consisting of RO 116-2617, RO116-0086 and RO 116-1148.
 34. The method of claim 29, wherein a partialagonist is administered.
 35. The method of claim 34, wherein the partialagonist is selected from the group consisting of LS65 0155, ML10302,RS67333, Prucalopride, and Cisapride.
 36. The method of claim 29,wherein the mammal is a human.